This invention relates to an apparatus and a method for the measurement of the heat generated in a chemical or biochemical reaction, by detecting and measuring a change in the conformation of a polymer transducer responsive to a heat change and being bound to the surface of a waveguide of an interferometer. The conformational change is detected by optical means and is compared with a control.
Assays involving biochemical reactions have hitherto generally required the use of a labelled reagent, for example a radioactive label or a fluorescent probe. Fluorescent labels have the disadvantage that they are generally bulky molecules that can change the structure and biological activity of the biochemical reagent that they are used to label. Radioactively labelling a molecule has the advantage that the molecule usually remains in its xe2x80x9cnativexe2x80x9d conformation. However, there are other drawbacks to this method, including: i) the radioactive decomposition of the molecule; ii) the radioactive label can generally only be inserted synthetically into the molecule; iii) if the radioactive label is added as part of a labelling reagent, it has the same disadvantage as fluorescent probes; and iv) handling and disposal of radioactive material. In those cases where no labelled reagent is used, it is usually necessary to develop methods for the separation of the reactants from products, prior to analysis of the amounts of reactant consumed, or product formed.
Ideally, a method that would use xe2x80x9cnativexe2x80x9d reagents and could monitor the progress of biochemical processes would be more suitable for the identification of enzyme inhibitors, antagonists, etc.
Essentially all biochemical and chemical reactions are associated with a change in enthalpy, and either take up or give off heat to the environment during the reaction. Since these reactions are typically performed in aqueous or organic solvent, the solvent changes temperature during the reaction. If this temperature change can be measured, then the rate and extent of the reaction can be measured directly. The ability to measure enthalpy changes through temperature changes in the solvent would obviate the need for labelling reagents, or the isolation of substrates or products to measure the extent of the reaction.
Microcalorimeters are useful for analytical measurements of biological or chemical reactions but are not currently useful in high throughput screening (HTS) applications for pharmaceutical screening purposes. Currently, microcalorimeters are capable of measuring enthalpy changes of 1 xcexcJ or greater and usually require test volumes of 1-1.5 ml. Most biochemical reactions have enthalpy changes of the order of magnitude xcx9c100 kJ/mol. In a typical biochemical assay volume of 100 xcexcl and a substrate concentration of the order of 1 nanomolar, the maximum amount of heat released by a sample and the corresponding temperature increase (assuming no heat is lost to the environment), is of the order 10xe2x88x928xc2x0 K. Thus, the temperature change associated with such assays is below the detection limits of current microcalorimeters.
Integrated optical Mach-Zehnder interferometers (IO-MZI) have been employed as sensors for the detection and measurement of biological and biochemical interactions.
For example, Brosinger et al (Sensors and Actuators, (1997), B 44, 350-355) describe an IO-MZI for use as an affinity sensor in which one branch of the device was coated with antigen and a phase change caused by specific interactions of immunoglobulins with the antigenic surface.
The present invention relates to an improved method and an apparatus for the measurement of heat changes in a chemical or biochemical reaction that increases the sensitivity of measurement compared with conventional methods, while concurrently reducing assay volumes and the quantity of reagents compared with those conventionally used in such assays. In addition, as discussed by Brosinger et al (loc cit), a major disadvantage of affinity biosensors is the difficulty in distinguishing unwanted non-specific reactions of protein-containing solutions with the sensor surface. The present invention overcomes this problem by preventing the sensor surface making contact with the sample.
In a first aspect of the invention there is provided a method for measuring a heat change in a reaction to be studied, the method comprising:
i) contacting a surface of a waveguide with a liquid including one or more components of said reaction to be studied said surface having a transducer responsive to a heat change in said reaction and being coated on and bound thereto;
ii) directing a beam of electromagnetic radiation through said waveguide in the absence and in the presence of an initiator of said reaction to produce a fringe pattern including a plurality of spaced light bands whose positions shift in response to changes in the heat of said reaction;
wherein the positional shifts of said light bands in said fringe pattern measured in the absence and in the presence of said initiator are used to calculate the heat change in said reaction.
In a second aspect of the invention, there is provided a method for measuring a heat change in a reaction to be studied, the method comprising:
i) contacting at least a portion of the surface of a sample waveguide and of a reference waveguide of an interferometer with a liquid including one or more components of said reaction, each said surface having a transducer responsive to a heat change in said reaction and being coated on and bound thereto;
ii) directing a beam of electromagnetic radiation simultaneously through said sample waveguide and said reference waveguide such that a fringe pattern is produced said fringe pattern including a plurality of spaced light bands whose positions shift in response to changes in the heat of said reaction;
iii) adding to said one or more components in contact with said sample waveguide an initiator of said reaction; and
iv) determining the heat change in said reaction by measuring the positional shifts of said light bands in said fringe pattern.
In a third aspect of the invention, there is provided an interferometric system, said system comprising a sample waveguide, a reference waveguide, and an outgoing waveguide;
wherein said sample and reference waveguides are formed of a material exhibiting an index of refraction, said sample and reference waveguides being joined at a first junction to an inlet waveguide and at a second junction to the outgoing waveguide such that electromagnetic radiation is allowed to pass simultaneously in parallel through said sample and reference waveguides;
characterised in that each of said sample waveguide and said reference waveguide comprises a surface that is coated with a transducer responsive to a heat change in a reaction and is bound thereto.
In a fourth aspect of the invention, there is provided use of an organic polymer or a biological macromolecule as a transducer for the measurement of a heat change generated in a chemical or a biochemical reaction to be studied.
The present invention therefore relates to the indirect determination of the heat of a reaction to be studied, suitably a biological or biochemical reaction, by monitoring a change in the conformation of molecules of a transducer that is in close proximity with a liquid reaction mixture, the transducer being coated onto and bound to the surface of the sample and reference waveguides of an interferometer. The change in conformation of the transducer molecules results in a change in the refractive index of the transducer, thereby producing a phase change in the electromagnetic radiation being transmitted through the waveguide.
Suitably, the interferometer is a Mach-Zehnder interferometer.
Suitably, the transducer is a material that is responsive to a heat change in the sample of the reaction to be studied, when the sample is in close proximity, or is in contact with, the transducer. The transducer is suitably a polymer, including an organic polymer or a biological macromolecule, which may be coated onto the surface of a region (the sensor region) of the sample and the reference waveguides. The polymeric transducer should be inert, that is, it must not interact chemically with the reagents taking part in the chemical or biochemical reaction under study.
Suitable polymers for use in the method according to the present invention include oligo- and polypeptides of greater than 10 amino acid monomer units, proteins, nucleic acids, oligo- and polynucleotides of greater than 10 nucleotide monomer units, and poly-olefinic compounds.
Preferred polymers for use in the method of the invention may be selected from the group consisting of: trypsin, myoglobin, ribonuclease A, fibrin, poly-L-glutamate, poly-xcex1-benzyl-L-glutamate, salmon DNA, sea urchin DNA, polyadenylic acid (double-stranded), poly adenylic acid (single-stranded), polyadenylic acid-polyuridylic acid, poly-methacrylic acid, poly-methyl methacrylate and poly-allylamine.
Suitably, the optical waveguides are formed of a material that exhibits a refractive index to the electromagnetic radiation that will pass through the waveguide. The design and fabrication of a suitable optical waveguide will be well known to the skilled person. See, for example, Heideman and Lambeck, Proc. IEEE/LEOS Symp. (1997), pp 29-32). Suitable materials for forming the waveguide are disclosed in U.S. Pat. No. 5,465,151. Suitable polymeric materials for forming the optical waveguide are disclosed in WO 01/06240 (Gharavi, A.), for example, polyimide-alkoxy sulphone diazo stilbene, polyimide-dialkylamino nitro azo and polyimide-dialkyl amino diazo. Examples of other materials that may be used for fabricating optical waveguides include, but are not limited to, silicon oxide, silicon nitride, gallium arsenide, as well as glass and other siliceous materials. Particularly preferred materials are selected from silicon oxide and silicon nitride.
The electromagnetic radiation that is transmitted through the waveguide is suitably light radiation in the visible to near-infra-red region, preferably in the visible region from 400 nm to 1000 nm. The visible light is suitably supplied by a laser or a laser diode source, for example a VCSEL at 850 nm or helium neon laser at 632.8 nm.
Suitably, the said one or more components of said reaction are components of a chemical or biochemical reaction. In principle, any chemical or biochemical reaction may be studied using the apparatus and method according to the present invention. Examples of reactions that may be studied include, but are not limited to, enzyme assays, immunoassays, hydridization assays (for example DNA/DNA, RNA/RNA, RNA/DNA), protein/nucleic acid interactions and protein/protein binding assays. The apparatus and method is suitable for the measurement of the heat generated or taken up in any chemical reaction (in which xcex94Hxe2x89xa00), for example catalytic conversions, adduct addition and cellular biochemical processes in which changes in the metabolic status of an organism may be accompanied by changes in the heat output from the organism.